Project Summary Iron active sites play key roles in O2 activation in biology. These are present in mononuclear non-heme Fe (MNHFe), binuclear non-heme Fe (BNHFe) and heme enzymes, which further divide into different subclasses (vide infra). We have developed a range of spectroscopic methods that enable the detailed study of the geometric and electronic structures of the FeII active sites and their oxygen intermediates, the coupling of electronic structure calculations to these spectroscopic data and the use of calculations supported by experiment in evaluating reaction coordinates. From our recent progress, formation of an FeIIIO2?- intermediate in the sulfur oxidizing/oxygenating enzymes (SOx) is stabilized by the substrate sulfur coordination, while for the Rieske dioxygenases (RDO) generation of this intermediate is uphill but its electrophilic attack is driven by a subsequent exergonic proton coupled electron transfer (PCET) from the Rieske center. The extradiol dioxygenases (EDO) also utilize an FeIIIO2?- reactive intermediate in electrophilic attack on a coordinated catecholate substrate. We now propose to complete the reaction coordinate for the EDOs and determine why these enzymes are selective in extradiol insertion and cleavage while the intradiol dioxygenases (IDO), which have a ferric active site, are selective for intradiol cleavage. For both the RDOs and EDOs, an FeIII(OOH) can also be formed but is relatively unreactive, while in a number of the BNHFe enzymes, peroxide intermediates are formed that are reactive in electrophilic chemistry. Our focus here is to understand the additional contributions of the second Fe in binuclear ferric centers that activate peroxide reactivity. For the a-ketoglutarate (aKG) and pterin dependent hydroxylases (PDH), an FeIV=O intermediate is formed and proceeds to react with substrate. Our research here focuses on the FeII/O2 reaction mechanisms for the generation of these FeIV=O species and how they enable selective hydroxylation, desaturation and electrophilic aromatic substitution (EAS). In the BNHFe enzyme soluble methane monooxygenase (sMMO), the peroxo biferric intermediate goes on to form a high valent Fe2IVO2 intermediate Q that H-atom abstracts (HAA) from the strong C-H bond of methane. Our focuses here are on determining why only this binuclear peroxo goes on to form a high valent intermediate, defining the geometric and electronic structure of Q and understanding the enhanced reactivity of this Fe2IVO2 intermediate relative to the MNHFeIV=O intermediates. An FeIV=O intermediate is also formed in the heme enzymes both with and without porphyrin oxidation and is also more reactive in the case of P450 than FeIV=O species in MNHFe sites. Our studies here are directed toward spectroscopically determining the effects of the porphyrin, its oxidation and of variation of the trans axial ligand on the FeIV=O bond; all topics thus far mostly addressed through calculations. Our studies should significantly contribute toward understanding the mechanisms of O2 activation and the reaction coordinates for the different subclasses of iron sites and generally toward understanding O2 activation at the superoxo, peroxo and oxo levels in biology and chemistry.